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Definitions

• the present invention relates to compounds, compositions and new methods for treating and/or preventing type II diabetes and related disorders and complications through uncoupling mitochondria.
• Type II diabetes is an adult-onset metabolic disease characterized by elevated plasma glucose concentration and insulin resistance of peripheral tissues. Type II diabetes inflicts about 20 million people in the US alone. If the hyperglycemic conditions in the type II diabetic patients are not intensively controlled pharmacologically, severe and sometimes fatal complications, such as cardiovascular diseases, heart attack, kidney failure, gastrointestinal diseases, gangrene, and blindness, can rapidly develop in patients. Obesity increases the risk of developing type II diabetes; however, obesity is not sufficient to cause type II diabetes, nor is obesity required for the development of type II diabetes. A majority of obese individuals do not develop diabetes; and type II diabetic patients are not always obese. Recent studies in type II diabetes research showed that it is not obesity per se that causes type II diabetes.
• Type II diabetes is the abnormal accumulation of lipid in liver and skeletal muscles that plays a causal role in the development of insulin resistance and hence type II diabetes. (Samuel V.T., et al., Lancet, 2010, 375:2267-77). Unfortunately, currently there are no cures for type II diabetes. Type II diabetic patients rely on pharmacotherapy for controlling the hyperglycemic symptom for the rest of their lives.
• the present invention is designed to improve diabetes therapy by providing compositions and methods for treatment and prevention of type II diabetes and obesity. It provides new clinical means of blood glucose control, by using the compound, 2', 5- dichloro-4'-nitro salicylic anilide and related compounds, which have mitochondrial uncoupling activities, to effectively reduce plasma glucose concentrations, increase insulin sensitivity, and reduce cellular energy efficiency, without the side effects and shortcomings of current methods of treatment.
• the family of 2-hydroxy-benzoic anilide compounds have been shown to be efficacious in treating and preventing type II diabetes when administered to mice. Acute administration of these compounds effectively reduces plasma glucose concentrations. Long-term oral administration increases insulin sensitivity, reduces fasting glucose and insulin levels. These outcomes result from mitochondrial uncoupling and disruption of the mitochondrial energy cycle. When analyzed on isolated mitochondria or in cell culture at concentrations comparable to plasma concentrations in vivo, these compounds uncouple mitochondria and stimulate oxidation of mitochondrial fuels without ATP production. The chemical modification that abolishes mitochondrial uncoupling activity of these compounds also abolishes the anti-diabetic effect. The safety of some members in this compound family in rodents and humans is well-established.
• the present invention provides use of a family of compounds in the treatment and prevention of type II diabetes and related disorders and complications, derived from the classes of compounds including, but not limited to, 2-hydroxy-benzoic anilide compounds, benzimidazoles, N-phenylanthranilates, phenylhydrazones, salicylic acids, acyldithiocarbazates, cumarines, and aromatic amines that have mitochondrial uncoupling activities.
• the present invention provides a method of treatment of type II diabetes and its symptoms, using the family of 2-hydroxy-benzoic anilide compounds or other mitochondrial uncouplers.
• the 2-hydroxy-benzoic anilide compounds suitable for use in the present invention include com ounds of formula (I):
• R 1 and R 11 are each independently hydrogen (H) or a protecting group that can be hydrolyzed in vivo to become hydrogen;
• R a and R b are each independently hydrogen or C]_-C alkyl
• the present invention provides a method of preventing metabolic and metabolism-related diseases or disorders, including, but not limited to, pre-type II diabetes, type II diabetes, obesity and obesity-related disorders and complications.
• the present invention provides a method of using these compounds to treat obesity, its symptoms and related conditions, including, but not limited to type II diabetes.
• the present invention provides a new approach for long-term chronic disease management by reducing plasma glucose.
• R 1 and R 11 are each independently hydrogen (H) or a protecting group that can be hydrolyzed in vivo to become hydrogen;
• R 2 through R 5 are independent, at least one of which is selected from the group consisting of -OH, halogen, -CN, -N0 2 , -CH(CH 3 ) 2 , -C(CH 3 ) 3 , and trihalo-methyl; and the rest of which are selected from -H, C 1-6 alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, Ci_ 6 haloalkyl, hydroxyCi-ealkyl, heteroaryl, and phenyl, wherein said heteroaryl or phenyl is optionally substituted with one to five substituents independently selected from CI, Br, F, CF 3 , and methoxy;
• R 6 through R 10 are independent, at least one of which is selected from the group consisting of -OH, halogen, -CN, -N0 2 , -CH(CH 3 ) 2 , -C(CH 3 ) 3 , and trihalo-methyl; and the rest of which are selected from -H, C 1-6 alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, Ci_ 6 haloalkyl, hydroxyCi-ealkyl, heteroaryl, and phenyl, wherein said heteroaryl or phenyl is optionally substituted with one to five substituents independently selected from CI, Br, F, CF 3 , and methoxy.
• the present invention provides compositions containing any of the compounds described above.
• the present invention provides use of the above-defined compounds in treatment or prevention of diabetes, in particular, type-II diabetes, and diabetes-related diseases or complications.
• the present invention provides use of the above-defined compounds or compositions for long-term management of diabetes and related diseases or complications.
• type II diabetes is caused by insulin resistance in peripheral tissues and is characterized by hyperglycemia, and reducing blood glucose is the most important therapeutic goal of treating type II diabetes.
• Mitochondria are critical organelles for glucose and lipid metabolism.
• the present invention uses 5-chloro-salicyl-(2-chloro- 4-nitro) anilide 2-aminoethanol salt (CSAA) as mitochondrial uncoupling agent for treatment of diabetic conditions.
• CSAA 5-chloro-salicyl-(2-chloro- 4-nitro) anilide 2-aminoethanol salt
• Intraperitoneal (LP.) injection of CSAA into db/db diabetic mice or high-fat diet induced pre-diabetic mice leads to effective reduction of plasma glucose levels. This is associated with increased AMPK (5' adenosine monophosphate- activated protein kinase) activity and increased glucose uptake in liver, skeletal muscles, and other tissues.
• AMPK 5' adenosine monophosphate- activated protein kinase
• the present invention provides a method of long-term disease management.
• Chronic oral treatment by adding CSAA into diet dramatically reduces fasting blood glucose levels in db/db diabetic mice.
• chronic feeding the high-fat diet induced pre-diabetic mice with CSAA greatly reduces fasting blood glucose and insulin levels, and increases insulin sensitivity.
• concentrations at which CSAA uncouples mitochondria in cultured cells are within the range of documented plasma CSAA levels after oral administration.
• changing the 2-OH group, which is essential for mitochondrial uncoupling activity, to 2-0-S0 2 H totally abolishes CSAA's efficacy in reducing plasma glucose concentrations.
• the chronic effect of CSAA on increasing insulin sensitivity is attributable to diminished lipid loads in liver or muscles.
• FIG. 1 illustrates acute treatment with 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt (CSAA) effectively reducing the blood glucose levels in the diabetic db/db mice and in the high-fat diet induced pre-diabetic mice.
• A Structure of CSAA. Blood glucose concentrations in (B) db/db mice or in (C) C57/B16 mice upon CSAA treatment. The db/db mice at the age of 6 weeks, or the C57/B16 wild type mice fed with high-fat diet (60% fat calorie) for 10 weeks starting from age of 5 weeks, were treated with the CSAA at dosage of 100 micro gram/mouse through LP. route.
• CSAA 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt
• mice were starved for 5 hours prior to CSAA injection.
• the blood glucose concentrations were measured at indicated time points after injection and normalized against the concentration at time 0 (untreated), which was set at 100%.
• FIG. 2 illustrates acute treatment with CSAA salt activating AMPK and increasing glucose uptake.
• A Levels of the phosphorylated AMPK in liver and muscle tissues upon CSAA treatment. C57/B16 mice were either treated with saline or saline containing CSAA LP. at the dosage of 100 microgram/mouse. 0, 2, or 4 hours later, mice were sacrificed and liver lysates were analyzed by immunoblotting to detect the levels of phosphorylated AMPK. The levels of the un-phosphorylated AMPK and RAN were also measured as controls.
• B Rates of glucose uptake in various tissues after CSAA treatment.
• mice were starved for 3 hours, followed by treatment with saline or saline containing CSAA LP. at the dosage of 100 microgram/mouse. 1.5 hours later, H-2-deoxyglucose (0.5 microcurrie/gram body weight) was injected LP. 30 minutes later, mice were treated with anesthesias and perfused with PBS. Mice were then sacrificed and tissue was extracted for measurement of H-2-deoxyglucose accumulation (normalized with tissue mass). UT, untreated; CSAA, CSAA treated; *, P ⁇ 0.05; **, P ⁇ 0.01. The data are representative results from two independent experiments.
• FIG. 3 illustrates two-week oral CSAA treatment reducing fasting blood glucose concentrations in db/db mice to almost normal levels.
• A fasting glucose concentrations
• B food uptake in db/db mice.
• the db/db mice at age of 6 weeks were fed with either normal AIN-93M food or with AIN-93M food containing 1500 ppm of CSAA salt for two weeks.
• the fasting blood glucose levels and food uptake of the mice were measured (normalized against grams of body weight).
• FIG. 4 illustrates chronic oral CSAA treatment reducing blood glucose and insulin levels in high-fat diet induced pre-diabetic mice.
• Normal C57/B16 mice were fed with high-fat diet (60% fat calorie) or high-fat diet containing 1500 ppm of CSAA for 8 weeks starting at age of 5 weeks.
• (C) daily food intake (between weeks 7 to 8 during high-fat diet feeding, normalized against grams of body weight) were measured.
• FIG. 5 illustrates chronic oral CSAA treatment increasing insulin sensitivity.
• FIG. 6 illustrates chronic oral CSAA treatment increasing AMPK activity and reduces liver lipid loads.
• A Phosphorylated AMPK levels in liver tissues of mice before and after oral CSAA treatment. The levels of the un-phosphorylated AMPK and RAN were also measured as controls.
• B Representative pictures of H & E stained liver tissues from mice either fed with high-fat diet alone (UT) or fed with high-fat diet containing 1500 ppm CSAA (CSAA) for 10 weeks. The white areas in hepatocytes are cells with high lipid content.
• FIGs. 7A and 7B depict the effect of chronic LP.
• 24 normal mice at the age of 8 months were fed with high-fat diet.
• Half of them (12 mice) were injected daily with CSAA via LP. route at the dosage of 100 ⁇ g/mouse (in 500 ⁇ PBS).
• the other 12 mice were injected with vehicle only (PBS).
• FIG. 8 illustrates mitochondrial uncoupling activities of CSAA, shown with isolated mammalian mitochondria (A) and in cultured mammalian cells (B) and (C).
• A Oxygen consumption chart of mitochondria isolated from mouse liver. Mitochondria, mitochondrial oxidative phosphorylation substrates, indicated inhibitors, and CSAA were added into the respiration chamber in the indicated order. Oxygen consumption was measured with an Oxygraph System.
• B) and (C) CSAA reduces mitochondrial membrane potential in mammalian cells. NIH-3T3 cells (-90% confluence) were treated with CSAA (B) at indicated final concentrations for 2 hours or (C) for indicated period of time at the concentration of 2 ⁇ . The cells were then stained with TMRE ( ⁇ ) for 15min to detect mitochondria membrane potential. After washed twice with PBS, cells were analyzed under microscope.
• FIG. 9 illustrates CSAA increases cellular oxygen consumption in the presence of oligomycin and does not increases cellular ATP levels.
• A Cellular oxygen consumptions were measured continuously for 120 minutes in cells upon treatment with DMSO (control), CSAA (1 ⁇ ), oligomycin (5 ⁇ / ⁇ 1), CSAA and oligomycin.
• CSAA dramatically increases cellular oxygen consumption even in the presence of oligomycin, indicating its mitochondrial uncoupling activity.
• B ATP concentrations were measured in cells treated with CSAA (1 ⁇ ) for indicated period of time. A total 20,000 cells under each condition were seeded and analyzed.
• FIG. 10 illustrates the diminished hypoglycemic effect of CSAA after converting 2-OH to 2-0-S0 2 H.
• A structure of the sulfite derivative of 5-chloro-salicyl-(2-chloro-4- nitro) anilide.
• the present invention relates to novel methods for treating, preventing, and alleviating the symptoms of, type II diabetes and diabetes-related disorders and complications.
• the family of 2-hydroxy-benzoic anilide compounds and derivatives can be administered as a means of blood-glucose and body- weight control by reducing plasma glucose concentration and cellular energy efficiency.
• the present invention provides a method of treating or preventing a metabolic disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a mitochondrial uncoupling agent.
• the mitochondrial uncoupling agent is selected from the group consisting of 2-hydroxy-benzoic anilide compounds, benzimidazoles, N-phenylanthranilates, phenylhydrazones, salicylic acids, acyldithiocarbazates, cumarines, and aromatic amines that have mitochondrial uncoupling activities, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I):
• R 1 and R 11 are each independently hydrogen (H) or a protecting group that can be hydrolyzed in vivo to become hydrogen;
• R through R are each independently selected from hydrogen, halogen, -CN,
• R a and R b are each independently hydrogen or CrC 6 alkyl
• R 2 through R 5 is not hydrogen
• at least one of R 6 through R 10 is not hydrogen
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I), wherein R 1 and R 11 are each independently hydrogen, -C(0)R 12 or -P(0)(OR 13 )R 14 , wherein: R 12 is hydrogen, -OR 15 , -NR a R b , Ci-C 2 o alkyl, C 2 -C 20 alkenyl, C 6 -C 10 aryl, or 5- to 10-membered heteroaryl;
• R 14 is -OR 15 , -NR a R b , Ci-C 20 alkyl, C 2 -C 20 alkenyl, C 6 -Ci 0 aryl, or 5- to 10- membered heteroaryl;
• R and R at each occurrence are independently hydrogen, C -C alkyl, C 6 -C 10 aryl, or benzyl;
• R a and R b are each independently hydrogen or CrC 6 alkyl
• any said alkyl or alkenyl is optionally substituted by one, two, or three substituents independently selected from hydroxyl, halo, C 1-4 alkoxy, and -C0 2 R 16 ; and wherein any said aryl, heteroaryl, and phenyl part of benzyl is optionally substituted by one to five substituents independently selected from C 1-4 alkyl, hydroxyl, halo, Ci-4 alkoxy, and -C0 2 R 16 ; and
• R 16 is hydrogen or CrC 6 alkyl.
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I), wherein:
• R 11 is hydrogen
• R is hydrogen or -C(0)R , wherein:
• R 12 is hydrogen, -OR 15 , -NR a R b , Ci-C 8 alkyl, C 2 -C 8 alkenyl, or phenyl;
• R 15 is hydrogen or CrC 6 alkyl
• R a and R b are each independently hydrogen or C]_-C alkyl.
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I), wherein R 1 is hydrogen, R a R b N-C(0)-, or an acyl group selected from the group consisting of:
• n 0, 1, 2, 3, 4, or 5;
• n is an integer from 1 to 200;
• R x at each occurrence is independently hydrogen or Ci-Cg alkyl
• R y at each occurrence is independently CrC 4 alkyl, halogen, hydroxyl, CrC 4 alkoxy, -N0 2 , -CN, or -C0 2 R x .
• R x is hydrogen; in another specific embodiment, m is 0; and in yet another specification embodiment, R y is -OH and m is 3. Therefore, the acyl groups of citric acid, succinic acid, fumaric acid, oxalic acid, gallic acid, and benzoic acid as protecting groups are encompassed.
• the mitochondrial uncoupling agent is a 2-
• R through R are each independently hydrogen, hydroxyl, halo, CrC 4 alkyl, CrC 4 alkoxy, CrC 4 haloalkyl, Cr C 4 haloalkoxy, and C -C acyloxy.
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I), wherein R 1 is hydrogen or acetyl; R 11
• R through R are each independently selected from the group consisting of hydrogen, hydroxyl, halo, nitro, and methyl.
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I), wherein R 1 is hydrogen or acetyl; R 11 is hydrogen; R 2 is hydrogen or methyl; R 3 is hydrogen; R 4 is CI or Br; R 5 is hydrogen; R 6 is hydrogen, -CI, -CH 3 , or -N0 2 ; R 7 is hydrogen or CI; R 8 is -H, -CI, or -N0 2 ; R 9 is H, CI, or Br; and R 10 is H or CI.
• formula (I) 2- hydroxy-benzoic anilide compound of formula (I), wherein R 1 is hydrogen or acetyl; R 11 is hydrogen; R 2 is hydrogen or methyl; R 3 is hydrogen; R 4 is CI or Br; R 5 is hydrogen; R 6 is hydrogen, -CI, -CH 3 , or -N0 2 ; R 7 is hydrogen or CI; R 8 is -H, -CI, or -N0 2
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I), wherein the compound is 2', 5- dichloro-4'-nitro salicylic anilide, or a pharmaceutically acceptable salt thereof.
• the mitochondrial uncoupling agent is a 2- hydroxy-benzoic anilide compound of formula (I), wherein the compound is 2', 5- dichloro-4'-nitro salicylic anilide 2-aminoethanol salt (CSSA).
• the metabolic disease or disorder is related to body-weight control.
• the metabolic diseases or disorder is selected from obesity, obesity-related complications, hypertension, cardiovascular disease, nephropathy, and neuropathy.
• the metabolic disease or disorder is related to elevated plasma glucose concentrations.
• the metabolic disease or disorder is type II diabetes, type I diabetes, or a related disease leading to hyperglycemia or insulin tolerance.
• the metabolic disease or disorder is type II diabetes or pre-type II diabetes.
• the metabolic disease or disorder is type I diabetes.
• the diabetes-related disease or disorder is selected from cardiovascular diseases, neurodegenerative disorders, atherosclerosis, hypertension, coronary heart diseases, cancer, alcoholic and non-alcoholic fatty liver diseases, dyslipidemia, nephropathy, retinopathy, neuropathy, diabetic heart failure, and cancer.
• the diabetes-related disease is a neurodegenerative disease.
• the neurodegenerative disease is amyotrophic lateral sclerosis, Parkinson's disease, or Alzheimer's disease.
• the mitochondrial uncoupling agent is used as a veterinarian drug to treat diabetes or a diabetes-associated disease, and the subject is a mammalian animal.
• the subject is a human.
• the mitochondrial uncoupling agent is administered in combination with a second anti-diabetic agent.
• the mitochondrial uncoupling agent is administered prior to administration of the second anti-diabetic agent.
• the mitochondrial uncoupling agent is administered concomitantly with administration of the second anti-diabetic agent.
• the mitochondrial uncoupling agent is administered subsequent to administration of the second anti-diabetic agent.
• the second anti-diabetic agent is selected from insulin, insulin analogs, sulfonylureas, biguanides, meglitinides, thiazolidinediones, alpha glucosidase inhibitors, GLP-1 agonists, DPP-4 inhibitors.
• the second anti-diabetic agent is metformin.
• the mitochondrial uncoupling agent is administered orally, intravenously, or intraperitoneally.
• the present invention provides a method for long-term disease management of a metabolic disease or disorder, comprising administering to a subject in need of such long-term management an effective amount of a mitochondrial uncoupling agent selected from 2-hydroxy-benzoic anilide compounds, benzimidazoles, N- phenylanthranilates, phenylhydrazones, salicylic acids, acyldithiocarbazates, cumarines, and aromatic amines that have mitochondrial uncoupling activities, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
• a mitochondrial uncoupling agent selected from 2-hydroxy-benzoic anilide compounds, benzimidazoles, N- phenylanthranilates, phenylhydrazones, salicylic acids, acyldithiocarbazates, cumarines, and aromatic amines that have mitochondrial uncoupling activities, or a pharmaceutically acceptable salt, solvate, or prodrug thereof.
• the metabolic disease or disorder is obesity, obesity-related complications.
• the metabolic disease or disorder is type II diabetes or diabetes-related complications.
• the present invention provides use of a mitochondrial uncoupling agent in manufacture of a medicament for treatment or prevention of type II diabetes, obesity, or related disorders or complications.
• R 1 and R 11 are each independently hydrogen (H) or a protecting group that hydrolyzed in vivo to become hydrogen;
• R 5 are independent, at least one of which is selected from the group consisting of -OH, halogen, -CN, -N0 2 , -CH(CH 3 ) 2 , -C(CH 3 ) 3 , and trihalo-methyl; and the rest of which are selected from -H, C 1-6 alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C . 6 haloalkyl, hydroxyCi-ealkyl, heteroaryl, and phenyl, wherein said heteroaryl or phenyl is optionally substituted with one to five substituents independently selected from CI, Br, F, CF 3 , and methoxy;
• R 6 through R 10 are independent, at least one of which is selected from the group consisting of -OH, halogen, -CN, -N0 2 , -CH(CH 3 ) 2 , -C(CH 3 ) 3 , and trihalo-methyl; and the rest of which are selected from -H, C 1-6 alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C . 6 haloalkyl, hydroxyCi-ealkyl, heteroaryl, and phenyl, wherein said heteroaryl or phenyl is optionally substituted with one to five substituents independently selected from CI, Br, F, CF 3 , and methoxy.
• n 0, 1, 2, 3, 4, or 5;
• n is an integer from 1 to 200;
• R x at each occurrence is independently hydrogen or Ci-Cg alkyl
• R y at each occurrence is independently CrC 4 alkyl, halogen, hydroxyl, CrC 4 alkoxy, -N0 2 , -CN, or -C0 2 R x .
• R x is hydrogen; in another specific embodiment, m is 0; and in yet another specification embodiment, R y is -OH and m is 3. Therefore, the acyl groups of citric acid, succinic acid, fumaric acid, oxalic acid, gallic acid, and benzoic acid as protecting groups are encompassed.
• R 1 is hydrogen or acetyl
• R 11 is hydrogen
• R 2 is hydrogen or methyl
• R 3 is hydrogen
• R 4 is CI or Br
• R 5 is hydrogen
• R 6 is hydrogen, -CI, -CH 3 , or -N0 2
• R 7 is hydrogen or CI
• R 8 is -H, -CI, -N0 2
• R 9 is H, CI or Br
• R 10 is H or CI.
• the present invention provides a composition for treatment or prevention of type II diabetes, obesity, a related disorder and complication, the composition comprising a compound of formula (I), or a pharmaceutically acceptable salt, solvate, or prodrug thereof, wherein:
• R 1 and R 11 are each independently hydrogen (H) or a protecting group that can be hydrolyzed in vivo to become hydrogen;
• R 2 through R 5 are independent, at least one of which is selected from the group consisting of -OH, halogen, -CN, -N0 2 , -CH(CH 3 ) 2 , -C(CH 3 ) 3 , and trihalo-methyl; and the rest of which are selected from -H, C 1-6 alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, Ci_ 6 haloalkyl, hydroxyCi-ealkyl, heteroaryl, and phenyl, wherein said heteroaryl or phenyl is optionally substituted with one to five substituents independently selected from CI, Br, F, CF 3 , and methoxy;
• R 6 through R 10 are independent, at least one of which is selected from the group consisting of -OH, halogen, -CN, -N0 2 , -CH(CH 3 ) 2 , -C(CH 3 ) 3 , and trihalo-methyl; and the rest of which are selected from -H, C 1-6 alkyl, Ci-ealkenyl, Ci-ealkynyl, Ci-ealkoxy, C . 6 haloalkyl, hydroxyCi-ealkyl, heteroaryl, and phenyl, wherein said heteroaryl or phenyl is optionally substituted with one to five substituents independently selected from CI, Br, F, CF 3 , and methoxy.
• the composition contains 2',5-dichloro-4'- nitro salicylic anilide 2-aminoethanol salt (CSSA).
• CSSA 2',5-dichloro-4'- nitro salicylic anilide 2-aminoethanol salt
• composition further contains a pharmaceutically acceptable carrier.
• the present invention provides a method of treating or preventing a metabolic disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a mitochondrial uncoupling agent or composition described above.
• the metabolic disease or disorder is type II diabetes, type I diabetes, or related diseases leading to hyperglycemia or insulin tolerance.
• the metabolic disease or disorder is type II diabetes.
• the diabetes-related disease or disorder is selected from cardiovascular diseases, neurodegenerative disorders, atherosclerosis, hypertension, coronary heart diseases, cancer, alcoholic and non-alcoholic fatty liver diseases, dyslipidemia, nephropathy, retinopathy, neuropathy, diabetic heart failure, and cancer.
• the diabetes-related disease is a
• the neurodegenerative disease is amyotrophic lateral sclerosis, Parkinson's disease, or Alzheimer's disease.
• the subject is a mammalian animal.
• the subject is a human.
• the mitochondrial uncoupling agent is administered in combination with a second anti-diabetic agent.
• the mitochondrial uncoupling agent is administered prior to administration of the second antidiabetic agent.
• the mitochondrial uncoupling agent is administered concomitantly with administration of the second antidiabetic agent.
• the mitochondrial uncoupling agent is administered subsequently to administration of the second antidiabetic agent.
• the second anti-diabetic agent is selected from insulin, insulin analogs, sulfonylureas, biguanides, meglitinides, thiazolidinediones, alpha glucosidase inhibitors, GLP-1 agonists, and DPP-4 inhibitors.
• the second anti-diabetic agent is metformin.
• the mitochondrial uncoupling agent is administered orally, intravenously, or intraperitoneally.
• the present invention provides use of a compound of formula (I) described above as a mitochondrial uncoupling agent in manufacture of a medicament for treatment of diabetes, obesity, or related disorders or complications.
• the present invention provides, among others, a method of treating and alleviating the symptoms of pre-type II diabetes (characterized by elevated blood glucose level) and complications of obesity or diabetes-related metabolic disorders, including, but not limited to, hypertension, cardiovascular diseases, nephropathy, and neuropathy. These diseases or disorders may be caused by dietary, environmental, medical and/or genetic factors.
• the method of the present invention can also be used for prevention of pre-type II diabetes and type II diabetes for a subject with risk factors including, but not limited to, obesity, dietary, and genetic predispositions and prevention of patients at risk from becoming obese and/or have obesity-related complications.
• the present invention provides a new approach for long-term chronic disease management and longevity management by reducing glucose levels in the blood.
• the present invention provides a method of treating or alleviating the symptoms of type II diabetes, using the family of 2-hydroxy-benzoic anilide compounds and derivatives or related compounds.
• Representative examples of 2-hydroxy-benzoic anilide compounds are set forth in Table 1.
• Mitochondria are organelles in cells that are at the center of glucose and fatty acid metabolic pathways. They are the place where beta-oxidation of free fatty acids, citric acid cycle, and oxidative phosphorylation occur.
• the net effect of beta-oxidation, citric acid cycle, and oxidative phosphorylation are oxidation of pyruvate (from glycolysis of glucose) and fatty acids to produce carbon dioxide, water and the chemical energy for generation of a proton gradient across mitochondrial inner membrane.
• the proton influx across the mitochondrial membrane Fo-Fi-ATPase drives the formation of ATP molecules.
• the proton gradient across mitochondrial membrane can be dissipated, a process called mitochondrial uncoupling, which causes a futile cycle of oxidation of lipids or pyruvate (from glucose) without generating ATP.
• Mitochondrial uncoupling can be induced by chemical uncouplers, for example, 2,4-dinitrophenol (DNP), which has various major side effects at higher doses, including causing hyperthermia.
• DNP 2,4-dinitrophenol
• this invention was designed to search for safe chemical mitochondrial uncouplers and evaluate their efficacy in treating type II diabetes.
• 5-chloro- salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt (CSAA), a salt form of an FDA approved anthelmintic drug whose mechanism of action is uncoupling mitochondria in parasites, is highly effective in reducing fasting blood glucose and insulin concentrations, increasing glucose uptake, increasing insulin sensitivity, and reducing liver lipid load. Its limited solubility would prevent or eliminate the side effects observed with DNP at high dosages. It is expected that the derivatives of this compound, their prodrugs, or other mitochondrial uncouplers that have limited solubility would have a similar efficacy and safety profile.
• DNP is, in fact, not an efficient mitochondrial uncoupler, which functions at mini molar concentrations.
• DNP has side effects that are not only associated with its uncoupling activity at high dosages, such as hyperthermia, but also it has side effects that are specific for DNP. Fortunately, mitochondrial uncoupling activity turns out to be not inherently associated with severe adverse effects.
• This invention demonstrates that CSAA is a much more efficacious mitochondrial uncoupler. It functions at high nano molar to low micro molar concentrations. What really sets CSAA apart from DNP is that CSAA has very limited solubility in aqueous solution.
• ROS mitochondrial reactive oxygen species
• alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.
• “Ci-Cio alkyl” or “Ci-io alkyl” (or alkylene) is intended to include Ci, C2, C3, C4, C5, C ⁇ , C7, Cg, Cg, and C10 alkyl groups.
• “C1-C6 alkyl” or “Ci-6 alkyl” denotes alkyl having 1 to 6 carbon atoms.
• Alkyl group can be unsubstituted or substituted with at least one hydrogen being replaced by another chemical group.
• alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t- butyl), and pentyl (e.g. , n-pentyl, isopentyl, neopentyl).
• Alkenyl is intended to include hydrocarbon chains of either straight or branched configuration having the specified number of carbon atoms and one or more, preferably one to three, carbon-carbon double bonds that may occur in any stable point along the chain.
• C2-C6 alkenyl or “C2-6 alkenyl” (or alkenylene), is intended to include C2, C3, C4, C5, and ⁇ alkenyl groups.
• alkenyl examples include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4- methyl- 3 -pentenyl .
• Alkynyl is intended to include hydrocarbon chains of either straight or branched configuration having one or more, preferably one to three, carbon-carbon triple bonds that may occur in any stable point along the chain.
• C2-C6 alkynyl is intended to include C2, C3, C4, C5, and Ce alkynyl groups; such as ethynyl, propynyl, butynyl, pentynyl, and hexynyl.
• alkoxy refers to an -O-alkyl group.
• C1-C6 alkoxy or “Ci-6 alkoxy” (or alkyloxy) is intended to include Ci, C2, C3, C4, C5, and Ce alkoxy groups.
• alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and t-butoxy.
• halo refers to fluoro, chloro, bromo, and iodo.
• Haloalkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with one or more halogens. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, and trichloromethyl.
• Haloalkoxy or "haloalkyloxy” represents a haloalkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge.
• C1-C6 haloalkoxy or "Ci-6 haloalkoxy”
• Ci Ci
• ⁇ haloalkoxy groups examples include, but are not limited to, trifluoromethoxy, trichloromethoxy, and 2,2,2-trifluoroethoxy.
• Aryl groups refer to monocyclic or polycyclic aromatic hydrocarbons, including, for example, phenyl, and naphthyl.
• C6-C10 aryl or “C6-10 aryl” refers to phenyl and naphthyl.
• aryl may be unsubstituted or substituted with 1 to 5 groups selected from -OH, -OCH3, -CI, -F, -Br, -I, -CN, -N0 2 , -NH 2 , -NH(CH 3 ), -N(CH 3 ) 2 , -CF 3 , -OCF3, -C(0)CH 3 , -SCH3, -S(0)CH 3 , -S(0) 2 CH 3 , -CH 3 , -CH 2 CH 3 , -C0 2 H, and -C0 2 CH 3 .
• benzyl refers to a methyl group on which one of the hydrogen atoms is replaced by a phenyl group, wherein said phenyl group may optionally be substituted by one to five, preferably one to three, substituents independently selected from methyl, trifluoromethyl (-CF 3 ), hydroxyl (-OH), methoxy (-OCH 3 ), halogen, cyano (-CN), nitro (-N0 2 ), -C0 2 Me, -C0 2 Et, and -C0 2 H.
• benzyl group include, but are not limited to, PhCH 2 -, 4-MeO-C 6 H 4 CH 2 -, 2,4,6-tri-methyl-C 6 H 2 CH 2 -, and 3,4-di-Cl-C 6 H 3 CH 2 -.
• heteroaryl is intended to mean stable monocyclic and polycyclic aromatic hydrocarbons that include at least one heteroatom ring member, such as sulfur, oxygen, or nitrogen.
• Heteroaryl groups include, without limitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrroyl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, and benzod
• Heteroaryl groups are substituted or unsubstituted.
• the nitrogen atom is substituted or unsubstituted (i.e., N or NR wherein R is H or another substituent, if defined).
• the nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N ⁇ 0 and S(0) p , wherein p is 0, 1 or 2).
• pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and/or other problem or complication, commensurate with a reasonable benefit/risk ratio.
• pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof.
• examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids.
• the pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
• such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic.
• inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric
• organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
• the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods.
• such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
• Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 18 th Edition, Mack Publishing Company, Easton, PA, 1990, the disclosure of which is hereby incorporated by reference.
• compounds of Formula (I) may have prodrug forms. Any compound that will be converted in vivo to provide the bioactive agent ⁇ i.e., a compound of Formula (I) is a prodrug within the scope and spirit of the invention.
• a prodrug within the scope and spirit of the invention.
• Various forms of prodrugs are well known in the art. For examples of such prodrug derivatives, see: a) Design of Prodrugs, edited by H. Bundgaard (Elsevier, 1985), and Methods in Enzymology, Vol. 112, at pp. 309-396, edited by K. Widder et al. (Academic Press, 1985);
• prodrugs Preparation of prodrugs is well known in the art and described in, for example, Medicinal Chemistry: Principles and Practice, F.D. King, ed., The Royal Society of Chemistry, Cambridge, UK, 1994; Hydrolysis in Drug and Prodrug Metabolism. Chemistry, Biochemistry and Enzymology, B. Testa, J. M. Mayer, VCHA and Wiley- VCH, Zurich, Switzerland, 2003; The Practice of Medicinal Chemistry, C. G. Wermuth, ed., Academic Press, San Diego, CA, 1999.
• the compounds of the present invention may be prepared by the exemplary processes described in relevant published literature procedures that are used by one skilled in the art.
• the 5-week old db/db mice and the C57/B16 mice were purchased from Jackson Laboratory and housed in the vivarium of UMDNJ-RWJMS. Starting at the age of 6 weeks the db/db mice were either fed with normal AIN-93M (Research Diet) or with AIN-93M diet containing 1500ppm CSAA. For the C57/B16 mice, at the age of 5 weeks, the mice were either fed with high-fat diet (60% fat calorie, Research Diet), or with high- fat diet containing 1500ppm CSAA. For acute LP.
• mice were starved for 5 hours, CSAA or the sulfite conjugated CSAA (customer synthesized by Provid Inc.) were dissolved in saline or saline containing 10% DMSO. A total volume of 100 micro liter solution (with or without 100 microgram CSAA or its derivative) was injected in each mouse.
• sets of mice were housed individually in metabolic cages (Nalgene) with free access to food and water. Food cups and food scattered in the runway to the cups were weighed daily to determine food intake.
• mice were sacrificed by decapitation, and the tissues of interesting were obtained.
• Blood glucose was determined using OneTouch UltraSmart blood glucose monitoring system (Lifescan), and the insulin levels were measured by ultra sensitive mouse insulin ELISA kit (Crystal Chem Inc.), following the manufacturer's instructions. Glucose tolerance assay and insulin tolerance assay
• mice were starved overnight and injected LP. with 20% glucose at a dose of 2 g/kg body weight. Blood was obtained from the tail at time points 0, 15, 30, 60, 90, and 120 min for glucose measurement.
• mice were starved for 5 h and injected LP. with 0.75U/kg body weight recombinant human insulin (Eli Lilly). Blood was obtained from the tail at time points 0, 15, 30, 60, 90, and 120 min for glucose measurement.
• Immunoblotting assays were carried out according to standard protocol.
• the sources of the antibodies are: AMPK antibody (Cell Signaling Technology), Thr-172- phosphorylated AMPK antibody (Cell Signaling Technology), Ran antibody (C-20, Santa Cruz).
• mice sacrificed by decapitation. Liver slices were fixed with buffered formalin (Surgipath Medical Industries, Inc.) and embedded in paraffin. Tissue slides were stained with hematoxylin and eosin (H&E) for detection of lipid droplets in tissue samples. Pictures were taken with a Universal Microscope Axioplan 2 imaging system (Carl Zeiss) with phase contrast objectives. Glucose uptake assay with H 2-deoxyglucose
• mice were starved for 3 hours, followed by treatment with saline or saline containing CSAA through LP. injection at the dosage of 100 microgram/mouse. 1.5 hours later, H-2-deoxyglucose (0.5 microcurrie/gram of body weight) was injected through LP. route. 30 minutes later, mice were treated with anesthetics and perfused with PBS. Mice were then sacrificed and tissue was extracted for measurement of H-2-deoxyglucose accumulation (normalized against tissue mass).
• Mitochondria were isolated from mouse liver. 1.0 mg of mitochondria in a volume of 0.9 ml of respiration buffer was analyzed for oxygen consumption in the presence of mitochondrial substrate as well as the various inhibitors with an Oxygraph System (Hansatech Instrument, Norfolk, UK). The final concentrations of the various chemicals added into the respiration buffer are as follows: succinate, 5 mM; ADP, 125 ⁇ ; oligomycin, 5 ⁇ g/ml; KCN, 2 mM)
• the NIH- 3T3 were culture to 90% confluence.
• the cells were then treated with CSAA at various concentrations and for various periods of time.
• the cells were then treated with TMRE (Tetramethylrhodamine ethyl ester perchlorate) to a final concentration of ⁇ , incubate for 15 minutes.
• the cells were then washed twice with PBS, and the pictures were taken under microscope.
• BDTM Oxygen Biosensor System was used to measure the cellular oxygen consumption.
• NIH-3T3 cells were cultured overnight to log phase, then seeded in oxygen biosensor 96-well plate at the density of 20000 cells per well in DMEM medium.
• Treatments were initiated by adding indicated drug CSAA (1 ⁇ ) or oligomycin (5 ⁇ g/ml), or both into the medium.
• Oxygen consumptions (decrease in oxygen concentrations) were indicated by generation of fluorescent signals which were initially quenched by oxygen.
• Cellular ATP concentrations were measured with the ENLITEN ® ATP Assay System and normalized against cell number.
• CSAA 5-chloro-salicyl-(2-chloro-4-nitro) anilide 2-aminoethanol salt
• AMP- activated kinase AMP- activated kinase
• Chronic oral treatment with CSAA reduces fasting blood glucose concentrations in db/db diabetic mice.
• a two week oral treatment of CSAA by feeding the mice with food containing CSAA
• the food intake rates between the two groups were not significantly different (Fig. 3B), which ruled out the possibility that CSAA may affect appetite and food uptake thereby causing the hypoglycemic effect.
• Chronic oral treatment with CSAA reduces fasting blood glucose and insulin concentrations in high-fat diet induced pre-diabetic mice.
• CSAA has no effect in food uptake in the mice (Fig. 4C). Moreover, the body weight and adiposity of the CSAA- treated mice were not different from the control mice (data not shown), again indicating that the anti-diabetic effects of CSAA were not mediated through reducing the degree of obesity. Together, these results indicate that CSAA can be highly effective in preventing diabetic conditions induced by high fat diet.
• Chronic oral CSAA treatment increases insulin sensitivity.
• the CSAA fed mice exhibited significantly increased insulin sensitivity as measured either by glucose tolerance assay or by insulin tolerance assay. These results indicate long-term CSAA treatment can increase insulin sensitivity. This effect may have contributed to the dramatically reduced fasting glucose and insulin levels observed in the Fig. 4.
• Example 6 Chronic oral CSAA treatment increases tissue AMPK activity and reduces liver lipid load.
• CSAA reduces fasting glucose concentration and improves insulin sensitivity
• oral CSAA treatment chronically elevated the AMPK activity in liver.
• oral CSAA dramatically reduced the lipid load in hepatocytes (Fig. 6B).
• hypoglycemic effect of CSAA may relate to its acute effect in reducing cellular ATP levels thus increasing glucose uptake, as well as to its long-term effect in reducing lipid accumulation in peripheral organs such as liver, which increases insulin sensitivity.
• Chronic CSAA treatment via LP. injection reduces high-fat induced weight gain.
• Those mice that were treated with CSAA via chronic oral administration as described from Fig. 2 to Fig. 6 exhibited significantly improved glycemic control, yet they did not differ from the untreated control mice in terms of body weight and adiposity. These results indicate that the anti-diabetic effects of CSAA in these mice were not mediated by reducing the levels of obesity.
• To increase the bio- availability of CSAA we performed chronic CSAA treatment via LP. injection and the effect of CSAA on high-fat diet induced body weight gain was examined.
• mice 24 normal mice at the age of 8 months were fed with high-fat diet. Half of them (12 mice) were treated with daily CSAA injections via LP. route at the dosage of 100 ⁇ g/mouse (in 500 ⁇ PBS). The other 12 mice were injected daily with vehicle only (PBS). The body weight was measured twice a week and the net body weight gain for each mouse was determined. The average weight gain in each group over the experimental period was plotted. As shown in Figure 7B, CSAA reduces the weight gain induced by high-fat diet. To rule out the possibility that CSAA affects body weight gain by reducing appetite, the food uptake rate of each mouse was also determined.
• Example 8 CSAA causes mitochondrial uncoupling at high nanomolar concentrations in cultured mammalian cells.
• Niclosamide the free base of CSAA, is an FDA approved anthelmintic drug. The mechanism of action of niclosamide is uncoupling mitochondria in roundworms and other parasites in intestine.
• Niclosamide is extremely insoluble in aqueous solution, which is probably responsible for its low systemic bioavailability and excellent safety profile.
• the water solubility of CSAA is about 30 to 50 fold higher than niclosamide fee base, with a maximal plasma concentration at around 0.75 -2.0 micromolar after oral administration (0.25 to 0.60 mg/L)
• a maximal plasma concentration at around 0.75 -2.0 micromolar after oral administration (0.25 to 0.60 mg/L)
• WHO/VBC/DS/8863 WORLD HEALTH ORGANIZATION FOOD AND AGRICULTURE ORGANIZATION, 1988. http://www.inchem.org/documents/pds/pds/pest63_e.htm).
• CSAA has mitochondrial uncoupling activity on mammalian mitochondria isolated from mouse liver.
• oligomycin which inhibits F o F ATPase
• CSAA still effectively promoted mitochondrial oxygen consumption, a feature that is unique to mitochondrial uncoupler.
• CSAA When analyzed with intact cells, as shown in Fig. 8B, CSAA exhibited activity in reducing mitochondrial membrane potential starting at the concentration of 500 nM. Full uncoupling of mitochondria could be seen at concentrations around 5 micromolar.
• the action of CSAA in dissipating mitochondrial membrane potential was rapid, and its effect could be seen as early as 5 minutes after CSAA application (Fig.
• CSAA stimulates cellular oxygen consumption with or without co-treatment of oligomycin and does not affect steady-state ATP concentrations in cells.
• CSAA uncouples mitochondria at low micro molar concentrations in living cells and to rule out the possibility that the reduction of mitochondrial membrane potential as observed in Fig. 8 is due to loss of mitochondrial integrity
• oxygen consumption of the intact cells upon treatment with CSAA in the presence or absence of oligomycin we measured oxygen consumption of the intact cells upon treatment with CSAA in the presence or absence of oligomycin.
• Fig. 9A CSAA dramatically stimulated cellular oxygen consumption, indicating the function of the mitochondrial electron transport chain was normal and was activated by CSAA.

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